INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com ISSN 2230 – 8407
Research Article
A DISCUSSION ON INDIAN INDIGENOUS LAL: ORLYSATE Deepak Bhattacharya*
Sri Radha Krishna, Kedar Gouri Road, Bhubaneswar, Odisha, India
Article Received on: 05/06/12 Revised on: 14/07/12 Approved for publication: 20/07/1
*Email: [email protected]
ABSTRACT
To discuss the use of LAL by using a reagent called ORLYSATE (LAL) –made from the (blue blood) hemolymph of the Orissan Horse Shoe Crab. Process technology for ORLYSATE has been developed in India {by this author}. It has been standardized at 10−15 and at 10−12, which are picogram and nanogram
sensitive, respectively {i.e., nano technology}. Endotoxin in pharmaceuticals could be assayed via agglutination in 1 hr and via chromogenic quantified route in 16 minutes. All orals, injectables and parentrals can be directly assayed. Residual endotoxin, contamination and status of sterilisation (LPS free) could similarly be assayed via elution, soak and sample method. Septicemia / Endotoximia can also be determined at clinical level. ORLYSATE can also be used to assay potable water, meat and other eatables. LAL / ORLYSATE is mandatorily required for determining quality control at production level apart from other uses an test kit for meningitis.By-products equally expensive e.g. Import substitution and Exportable Antibiotic, Glycoprotiens and Immunoglobulins.
KEY WORDS: Horse Shoe Crab, India-Odisha, LAL, Orlystae, Numerous Species, Nano-Technology.
INTRODUCTION
Of the long stretch of the eastern shore board of India, the northern shores of Odisha and the southern shores of West Bengal is home to the horse shoe crabs. It is a living fossil, in other words it has not undergone any physiological change since the Precambrian era- (390 x 160 years) or 350-450 million years. From its blood Lysate is made. Although it is called a crab, Zoologist place it in the scorpion family. It is an amphibian, very docile and without any instinct of offence. Interestingly its blood has the colour of human pus. As because its oxygen carrier is copper {as in snails/zancus
pyurm, etc}, the serum turns blue only on oxygenation
{instead of amber – as with anthropomorphic blood}. This crab is found only in a small pocket off the eastern coast of USA, India, Philippines, China and Japan, Rest of the world does not have it. While the North American species is Limulus; the Japanese and the Indian are Tachypleus and the Sino land species is Carcinocorpus. Lysate from the Japanese (Asian) and the American Crabs indicate/demonstrate identical gelling activity to purified lippolysaccharides (control standards). Due to drastic reduction in Crab census/population along the American coast. US companies have set up plants in China and elsewhere in Asia (except India). They are said to be eyeing the Indian source.
In this discussion we present the applied aspects. The term ORLYSATE is the designated Trade Mark call name of this indigenously invented product, and the technology. It has also been done natively. The said is now poised for an application with Patent authority. Few aspects of the Indian ecology and the available species spectrum are also presented. Apart from the species Tachypleus Gigas,
evidence of few other species are presented. This makes India as the sole place that has numerous species. Earlier, in this journal Sushruta and Anubha1 have presented an overview. However, it does not mention anything about the India-effort story, nor about the real-time diagnostic application. This report therefore offers an exiting opportunity to scholars to work on each of these species and about the yield, sensitivity, seasonality aspects from pharmaco-biochemical aspects. Between 1970-1982 research for the possibility of tapping this source for an alternative pyrogen test route was undertaken extensively. This route came to be called as
Lysate test route or LAL test. L-Limulus indicated the Crab, A-Amebocyte indicated the lymph cell containing platelet factor and L-Lysate denotes a lysed preparation.
THE ORLYSATE
LAL- Limulus Amebocyte Lysate - is an enzyme fraction stored in lyophilised form. LAL or lysate as it is more popularly called, detects picograms of endotoxin. We have been able to develop famtogram sensitive LAL. Endotoxins are lippolysaccharides released from the cell walls of the gram negative bacteria. Elimination of endotoxin in pharmaceutical preparations is of paramount importance. Degree of elimination is determined by assaying (testing) such pharmaceuticals for residual endotoxin or pyrogen. Traditionally it has been done by the rabbit pyrogen route i.e. by injecting a rabbit such formulations and then reading febrile reactions if any. Rabbit route or animal route is a biological test and is done in-vivo. Therefore there are limitations galore.
Scientists have been on the constant lookout for specific, failsafe and more sensitive route. In 1956 F.B. Bang2 observed that gram negative infection caused massive vascular coagulation in the Horse Shoe Crab. Ever since then, the blood of this crab has been researched in depth and finally established that the blood of the Horse Shoe Crab contains enzymes which gels and forms a clot when exposed to endotoxin of nano quantities (10−15 part of a gram). Due to its ultra sensitivity, reproducibility, failsafe character, speed and being a chemical in-vitro test, the US Pharmacopoeia made Lysate test mandatory. Others followed. The first non-resident Indian to have got involved with LAL research was Rajiva Nandan3. The first native Indian pharmaceutical company to have funded LAL research in India was M/s Unichem with NIO-CSIR as the first national level resource partner4. Even then, LAL remained elusive.
base. Following the US5,6, Japan and the European community have similarly recognised it7. The Indian Pharmacopoeia has also recognised it and made LAL test mandatory for-5 pharmaceuticals (1) Sterile water (2) Large Volume Parenterals (3) Analgesics (4) Anti-pyretics and (5) Diazepam. These apart, there are numerous other uses8,9, including attempts to use of LAL in clinical diagnosis viz.,sepsis10 and in urinary tract infection11. Use in parentral and intrathecals constitutes the most12.
With Lysate, Radio pharmaceuticals, Surgical devices, Attachments, Infusion and Transfusion sets of every description can be assayed for a clear YES or NO to indicate presence of endotoxin. This apart it is being employed to numerically quantify such (residual) endotoxin. All this takes an analyst (qualify controller) 60 minutes to 15 minutes only.
TABLE – I: The Two Routes Compare Thus13,14 Rabbit (Route) (Orlysate / Lysate Route)
1 In Vivo In-Vitro
2 Biological Chemical
3 Subjective Objective(says yes or No)
4 Debatable Clear Visible to naked eye
5 Long Preparation Very short
6 In process test not possible Designed for such work
7 USP de-Recognised USP Recognised
8 Rabbit colony/Animal Required
Not Required
9 Expensive Inexpensive
10 Time 4-12 hours 16mnts to 1 Hour only.
Lysate test is done by three routes: (a) Gel clot method (b) Turbidimetric (c) Chromogenic. Of late, the turbid route is being used by the leading US manufacturing firms. It entails use of much less quantities of the expensive LAL and is also provides ease of quantification in one go via cartridge. Indian Pharmacopoeia has recognised only Gel Clot Route15. According to this indigenous technology, the Indian Horse Shoe Crab has been turned into a donor. It donates approximately 25-30 ml of blood at an interval of 90 days. The crab is caught manually and is released back to sea post hemolymph tapping. From this volume 2-5 ml of ORLYSATE (Indigenous Lysate) is got, which yields 5-10 test per ml. of 0.06 IU (0.0125) ng = 12.5pg sensitivity. An endotoxin pass/fail limit of 0.5 IU/ml for pharmaceuticals is equivalent to the rabbit threshold pyrogen dose of 0.1 ng/kg when test solution is administered at 10 ml/kg. Orlysate is many times more sensitive as compared to rabbit route16. After Lysate is taken out {expensive process} from the blue blood the following by-products can be made from the said to be waste: (a) Hemocynine (glycoprotein) (b) Polymexin-B-(group OM permiabiliser/ bacteriostatic). We have also isolated (b) and have named it as ORLISIN. Both equally important in human and veterinary health services and are very expensive. Test volume being 0.1 ml or 0.025 ml. Present day cost being very high, there is tremendous demand for it.
DIAGONISTIC KIT
Meningitis test kit can be developed. It yields result in 16 minutes to 1 hour only. However, it is currently not relevant from commercial/industrial perspectives.
GEL CLOT METHOD
One hundred microliter (0.1 ml) of product/sample solution is taken in a pyrogen free 75 x 10 mm glass tube, add Orlysate of equal quantity, mix gently, avoid foam, incubate undisturbed for 60 minutes. A firm gel indicates presence of
pyrogen in product-called a positive (+ve) test. Absence or weak gel indicates No pyrogen (-ve) test result. A firm gel does not break when assay tube is inverted 1800.
Endotoxin can be detected in all types of (human and veterinary) surgical devices, pharmaceuticals, radio pharmaceuticals, blood and plasma products, packed food, meat, beverage and water. Moreover endotoxin can be quantified (which can not be done by the existing rabbit pyrogen route).
The LAL being a poly-peptide {labile enzyme} and the LPS {endotoxin} being a highly stable quasi srystaline, produce a limited proteolysis of the enzyme. Therefore, structure and function of the binding protein of the LPS is crucial. And, endotoxin has been held {extensively debated} as having variable affinity for the walls of the lab-wares. Among, the gamut of the LPS, that of the E-Coli has been found as the most suitable. It has been adopted and used worldwide {mostly strain 555:5B}. Hence lysate science is very interesting and oppertunity full.
QUANTIFICATION
Every sample is tested in series, diluted two fold and assayed with Orlysate of known (labeled) sensitivity to reach a point of dilution beyond which no more gel forms. This is known as ‘end point dilution’ (EPD). Antilog of the geometric mean end point of a replicate series yields the EPD value :
Endotoxin in sample = Orlysate sensitivity x EPD.
Where interference occurs due to due to high concentration of the formulation, a firm gel does not form, a maximum valid dilution factor (MVD) is taken - that is the product has to be assayed within the (MVD) factor.
ExP Or Endotoxin limit x Potency of Product MVD = L Orlysate Sensitivity
MVD = Formulations that have official endotoxin limits stated in the monograph now can be assayed to determine the endotoxin level by the above formula-and where no official limits are available a minimum valid concentration (MVC) factor has to be determined.
L x M MVC = K
L = Orlysate sensitivity. M = Rabbit Dose/Kg/hr. K = Endotoxin limit/kg. (1) Non Intrathecals 5.0 IU/kg. (2) Intrathecals 0.2 IU/Kg. P = Potency of Product. Now a MVC can be calculated. ___P___
MVD for Unknown Products = MVC
Replicating test series, taking their log value and calculating a mean of such EPD values, the GMEP is arrived at which when multiplied with labeled sensitivity of Orlysate yields the amount of endotoxin/unknown level of endotoxin. The labeled sensitivity can also be verified by running a number of series test with fold diluted control standard endotoxin CSE (USP). The antilog of geometric mean of end point values is the precise sensitivity factor of the reagent (Orlysate). It nearly matches the label. This precision is the backbone of the quantitative system.
END POINT
necessitates determination of an average - to pin point Lysate sensitivity termed GMEP.
GEOMETRIC MEAN END POINT AND LYSATE SENSITIVITY
Endotoxin ORLYSATE Dilution Series in water 0.5 IU;0.25 IU ; 0.5 IU 0.1 ml 0.1 ml + - -
0.1 ml 0.1 ml + + - 0.1 ml 0.1 ml + - -
End points marked in boxes. End Points Log 10
0.5 - 0.30103 0.25 - 0.60200 0.5 - 0.30103
- 1.20406 3 = - 0.4013538
Antilog of 0.4013538 = 0.3968 IU/ml or 0.4 or Orlysate Sensitivity = 0.4 IU/ml.
One half to two times variation from labeled Sensitivity is accepted by USP.
EPD = end point dilution. PPC = positive product control.
DETERMINNING ENDOTOX IN AN SAMPLE / PRODUCT
Step -:
1) Test serial two fold dilution of said sample to reach an end point.
2) Calculate geometric mean end point (GMEP).
3) Multiply - mean of GMEP with Lysate Sensitivity as printed on Lysate vial. OR calculated sensitivity.
OR =
Lysate sensitivity X end point dilution (GMEPD) Example
Sample to be diluted and assayed
Replicate 2 Fold 4 Fold 8 Fold 16 Fold 32 Fold Series -1 + + - - -
Series -2 + + + - - Result
End Point IU/ml Log 10 of end points Series -1 4 Fold 0.25 - 0.6020599 Series -2 8 Fold 0.125 - 0.9030899 GMEP = 1.5051499 2 = 0.7525749 Antilog 10 of GMEP (0.1767767) 1 IU 0.1767767 = 5.6 Fold. Endotoxin in sample =
Lysate sensitivity X end point dilution or = 0.4 x 5.6 = 2.24 IU/ml.
Thus with ORLYSATE, the manufacturer / analyst / can quantify endotoxin in their product by such simple method. ORLYSATE is suitable for most of the concentrated products for BET and quantification test through gel clot route. Higher the Lysate sensitivity greater will be the end point dilution. Greater the end point dilution (EPD) easier it will be for Lysate to over come product/sample inhibition (if any), endotoxin test and quantification of concentrated products. It also means concentrated products will yield higher end points dilution. AND normally require a greater Lysate sensitivity . Higher EPD is also indicative of higher endotoxin concentration specially in non concentrated products. Therefore relevance of MVD.
We however have incorporated inhibition remover into ORLYSATE and as well have an separate additive. All this makes Orlysate a nano technology product.
MAXIMUM VALID DILUTION - MVD
Many concentrated drug products interfere in the formation of a firm gel in the gel clot route. Dose dependent endotoxin limit as suggested by Industry and proposed formulas for determining MVD factor for such products is :-
FOR PRODUCTS WITH OFFICIAL ENDOTOXIN LIMITS
MVD E x P = Endotoxin limit x Potency of product L = Sensitivity of Lysate.
E = Endotoxin limit of the product
P = Labeled potency of product - on expiration date. L = Sensitivity of Lysate.
Example
E = 0.5 IU. P = 2 mg/ ml. L = 0.4 (as calculated) MVD = 0.5 X 2 mg / ml = 2.5 fold
0.4 IU / ml
FOR PRODUCTS WITH NO OFFICIAL LIMITS MVC = (Minimum valid concentration)
L x M K
L = Lysate sensitivity as on Label. IU/ml.
M = Rabbit dose/Kg that would be administered in one hour period excepting pediatric and Radio Pharmaceuticals, Where pediatric dose is available it shall be the
unit for calculation.
K = a) 5.0 IU/kg for parenterals not injected through intrathecal route.
b) 0.2 IU/kg for intrathecal route administered drugs. INDICATORS
1. Product must yield a clear assay result within calculated MVD ; else , product not suitable for BET by Lysate route. 2. Product EPD exceeding MVD is concrete evidence of concentration of endotoxin in excess of limit stated in monograph.
CHROMOGENIC METHOD
To 750μl of synthetic substrate # 50μl of catalyst cum accelerator is added, to which 20 – 50μl of sample to be tested is added. To this 20 – 50μl of Orlysate [LAL] is added , mixed gently and well, incubated at 370C for 15 minutes. At end 100 – 160μl of Glacial acetic acid is added to terminate the process .
This reaction releases p- Nitroaniline (yellow chrome) which is measured spectro-photometrically. The digital figure is correlated to a standard to instantly arrive at the amount of pyrogen / endotoxin present in the test sample.
Total volume adds up to 1ml AND time is reduced to
¼ of Gel clot method and is more specific in quantitative and absolute terms AND consumption of LAL is reduced drastically. Chromogenic Orlysate has been developed.
# Nt – Boc-Val- Leu-Gly-Arg-pNA; BZ- Ile-Glu-Arg-pNA . TURBIDEMETRIC METHOD
concept of turbidity is that when ORLYSATE is exposed to endotoxins, it reacts making the solution turbid {which is otherwise clear} within 15minutes. Permits shake. The intensity of the pass/fail turbidness {relative opacity} is pegged at pre-calibrated transmittance = sensitivity. The benefit of this route is that it requires less {i} LAL {ii} quick results {iii} fixed readings of pass/fail {iv} accurate reproducibility {v} less space {vi} cartridge possible. Turbidity reading desk-top devices can be made for products that have near similar transparency \ opaque to IR. This apart,
‘micro methods’ have also been tried long ago and which has now been commercially adopted in the west (see Ref. No. 21).
ORLYSATE TEST FOR PLASMA
Dilute plasma in 1/10 Ratio in Orlyqua (pyrogen free water), heat for 5-7 minutes at 75 degree and assay with ORLYSATE. All Blood samples to be assayed should be free of anticoagulants and of all external additives17.
TEST FOR SURGICAL DEVICES:
Surgical devices and attachments be dipped overnight around 30-37 C in pyrogen free water in pyrogen free container. An aliquot be assayed to determine residual pyrogen in normal sterilisation process. Container water be mixed thoroughly with pyrogen free stirrer before aliquot is taken18.
USP - REQUIREMENTS
(a) Lysate sensitivity / validation test ( Not less than 0.5 IU/ml).
(b) Inhibition / enhancement testing.
i - Devices of each materiel type, make and manufacturing process should be tested.
ii- Devices of common chemical formulation to be grouped and assayed.
iii- At least three production lots of each product type should be tested.
iv- Recommended quantities Lot sizes 2 Under 30
3 30 - 100
3 Above 100 ( Max.10 devices per lot )
The process of preparing an elute / extract for pyrogen or inhibition/enhancement testing may vary for each device. Some medical devices can be flushed, some may have to be immersed in the non-pyrogenic rinse solution, while others may be tested by disassembling or by cutting the device into pieces prior to extraction by immersion. In general, for devices being flushed, the non-pyrogenic rinse solution should be held in the fluid pathway for one hour at room temperature (above 180C); effluents should be combined. If a device is to undergo extraction, a minimum extraction time should be 15 minutes at 370C, one hour at room temperature (above 180C) or other demonstrated equivalent conditions. RINSE VOLUME GUIDELINES:
a. Each of the 10 test units should be rinsed with 40 ml of pyrogen free water.
b. For unusually small or large devices, the surface area of the device which comes in contact with the patient maybe used as an adjustment factor in selecting the rinsing or extracting volume.
The endotoxin limit can be adjusted accordingly.
The rinsing scheme should not result in a greater dilution of endotoxin than used in USP rabbit pyrogen of transfusion and infusion assemblies. For inhibition/ enhancement testing, both the rinsing/ extraction solution and the device elute /
extract should be assayed as prescribed for test in gel clot route.
PYROGEN LIMITS FOR MEDICAL DEVICES USP. i. Devices that contact cerebrospinal fluid - 0.06 IU/ml. ii. Devices that do not contact cerebrospinal fluid 0.5 IU / ml. Ideally choose the product with the largest surface area that contacts body or fluid being administered. IP has yet not recognised this requirement.
I P REQUIREMENTS
Indian Pharmacopoeia has been amended and gel clot test has been made mandatory for 1 – Diazepam; 2 – Anti-pyretics; 3 – Analgesics; and 5 - Large Volume parentrals. The above constitutes huge domestic market.
CAUTION
(1) Certain products may enhance ORLYSATE (Lysate) sensitivity and may give false positive. We recommend repeated Lysate test AND parallel Rabbit pyrogen test in such case. Ways and means to overcome too have been invented. (2) While Reconstituting control standard endotoxin and making fold dilutions thereof vortex vigorously for minimum 2 minutes every time. Fold dilute in cilliconised glassware or polystyrene containers. Glass absorbs endotoxin thereby weakening it. May give erroneous result. Vortex thoroughly before assay. Follow suppliers instruction meticulously OR OR use low potency CSE pack reconstituting fresh before each test program. Inhibition remover has also been developed.
(3) ORLYSATE turns brown when denatured.
(4) The standard curves differ for glassware and for plastic wares {higher}.
UNIQUE HABITAT CUM STOCK POOL
Fig. 5a is that of the dissection exercise being carried out by a multi-disciplinary team.
Fig. 5b is that of a dissected adult crab. It shows that the inside view of the ventral side. It is full loaded with eggs. The suggested fecundity is indeed very high {as compared to other amphibians}. It is a Limulus Spp. It also shows that it has paired oviducts.
Fig. 6 is that of a microscopic view of the blue blood. It shows the platelets are oval, measure 20μmt and the granulocytes contained inside.
Fig 7 to 9 are that of from the underwater. When compared with the feature as in Fig.-1019, they show various features of the various species20. Particularly the 2nd prosom appendages {all 4 species} distinctly match. This prima-facie suggest that the entire domain of morphological and species based studies relating to the Horse shoe crab offers unprecedented opportunity for the aspiring scholar and for the enterprising entrepreneur.
Moreover, There are numerous manufacturers21. Inter-make differ22. Pyrogens are also expressed by viruses and fungis and as well by gram+ve bacteriums. ORLYSATE can detect all. The challenges have been overcome and is poised for the opportunities.
CURRENT WORK FORTFOLIO
The current research portfolio includes {i} inhibition remover {ii} falls positive and falls negative generator – to aid the drug inspection and regulatory authorities {iii} a critical technology based fractionator. LAL has also been held as
LAL/Orlysate {vii} collected more field evidence of the variability in spp.. i.e., wider spectrum of raw material sources. All this will help all the concerned (augers well for the nation).
DISCUSSION CUM CONCLUSION
LAL has no direct medicine efficacy. Various media reports and claims by various persons have been made indicating that LAL as a panacea. All claims of its having uses in arthritis {other metabolic disorders} and cancer are baseless, and are done for dubious reasons. Indigenous Lysate when commercially made available will go a long way to ensure better quality standards of Indian pharmaceuticals, save foreign exchange as well. Over a period of time Lysate test is likely to find place in the curriculum of various medical sciences.
There are numerous marine species that have blue blood with the LAL moiety. However, non are in such volumes as that it can be used to make test kits. In Odisha, there are as many as 3 species –if not 4. This offers unprecedented scope for the scholars of pharmacology and medicinal chemistry to peruse for higher attainments. It also offers scope to the related industry.
This apart, some efforts have been made to develop assay reagents and methods using anthropogenic whole blood and or monocytes – as alternative to LAL. This is proving to be drug item/compound specific and takes more than 24hrs. Therefore, LAL has made a come back. Indigenous LAL technology which is a Nano Technology is now available in India and is ready for commercialization and also for advanced research.
REFERENCES
1 - Mulay S. and Khale A. {2012}, An Overview of Limulus Amebocyte Lysate, International Research Journal of Pharmacy, 2011;2(4):67-71. 2 - Bang Fredrick B, {1956}, A Bacterial Disease of Limulus Polyphemus,
Bull. Jhon Hop Hospital, Vol.98, 1956. pp.325-
3 – Nandan Rajiv and Brown David, {1977}, An Improved in-vitro Pyrogen Test : To Detect Picograms of Endotoxin Contamination in Intravenous Fluids Using Limulus Amebocyte Lysate, Jor of Lab Clin Med, 04-1977, Vol.89, No.4, pp.910-18.
4 – It was 1992-96 period, Mr. Chitta Behera of Project Swaraj, a NGO of Cuttack, Odisha, used to project author’s horse shoe crab ponds and other assets, to all concerned.
5 - US Dept. of Health and Human Services, Public Health Services ,{1987}, FDA- Guideline on Validation of the LAL test as an End Product Endotoxin Test for Human and Animal Parenteral Drugs, Biological Products and Medical Devices – 12/ 1987.
6 – Wachtel E. Ruth and Tsuji Kiyoshi, {1977}, Comparison of Limulus Amebocyte Lysates and Correlation with the United States Pharmacopeial Pyrogen Test, Applied and Environmental Microbiology, June 1977, Vol.33, No.6, pp. 1265-69.
7 – Noordwijk van J and Jong de Y. {1976}, Comparison of Limulus Test for Endotoxin with Rabbit test for Pyrogens of the European Pharmacopeia, Journal of Biological Standardisation, Vol.4, pp. 131-39.
8 – Jorgensen H. J., Lee, J.C., Alexander, A.G., and Wolf W.H. {1979}, Comparison of Limulus Assay, Standard Plate Count and Environmental Microbiology, Applied and Environmental Microbiology, Vol.37, pp. 928-31.
9 – Jay M., Slavoljub M., Alexa L., Shereda and Covington V.H., {1979}, Determining Endotoxin Content of Ground Beef by the Limulus Lysate test as a Rapid Indicator of Microbial Quality, Applied and Environmental Microbiology, Vol.38, No.5, pp. 885-90.
10 – Levin Jack, Poore E.T, Neil P, Zauber B.A, and Oser S Ronald, {1970}, Detection of Endotoxin in the Blood of Patients with Sepsis Due to Gram Negative Bacteria, The New England Journal of Medicine, Dec-1979, Vl.283, No.24, pp.1313-16.
11 – Jorgensen H. J, Carvajal H.F, Chipps B.E, and Smith R.F, {1973}, Rapid Detection of Gram Negative Bacteriuria by use of the Limulus Endotoxin Assay, Applied Microbiology, July 1973, Vol.26, No.1, pp. 38-42.
12 – Weary M. and Baker B. {1977}, Utilisation of Limulus Amebocyte Lysate Test for Pyrogen Testing Large Volume Parentrals, Administration Sets, and Medical Devices, Bulletin of The Parentral Drug Association, Vol. 34. No.3, pp.127-35.
13- Cooper F. James, Levin Jack, and Wagner N. Henry, Quantitative Comparison of in-vitro and in-vivo methods for the detection of endotoxin, Jor of Lab Clin Med, 07-1971, Vol.78, No.1, pp.138-48. 14 – Kothari Gunavanthi, {1996}, LAL Testing for Endotoxins, Express
Pharma Pulse, Nov. 26, pp.64-68.
15 - Director Central Drug Lab, Kolkata had sought the opinion and advice of the inventor pre amendment exercise (which was tendered in writing; adopted), 1994 and again 1995.
16 – Eibert J Jr., {1972}, Crabs vrs Rabbits, Bulletin of The Parentral Drug Association, Vol. 26, No.6, pp.253-260.
17 - Bhattacharya D {1995} All India Medical Conference, Bhubaneswar, Dec. 1995.
18 –Bhattacharya, D. {2001} International Rural Surgeon’s Conference, Puri-marine drive.
19 – Sekiguchi, K. and T. Nakamura, {1979}, "Ecology of the extant horseshoe crabs." In Biomedical Applications of the Horseshoe Crabs (Limulidae), edited by E. Cohen, 37–45. New York: Alan Liss, Inc., 1979.
20 - http://www.horseshoecrab.org/rsrch/rsrch_s.html 21 – Endosafe Times, {2007}, Vol.13. No.2, Nov, pp. 1-7.
22 – Christine W. Twohy M. Neirman L. and Duran, P.A, {1983}, Comparison of Limulus Amebocyte Lysate from Different Manufacturers, Journal of the Parental Science and Technology, Vol.37, No.3, pp.93-96.
23 – Elin, J. R. and Wolff, S.M. , {1973}, Nonspecificity of the Limulus Lysate Test : Positive Reactions with Polynucleotides and Proteins, The Jor of Infectious Disease, Vol.128, No.3, 09-1973, pp. 349-52.
Fig. – 1 Dorsal view of a adult Limulus Polyphemus.
Fig. – 3 Gel Clot Test at 100 μml : 100μml assay. Shows white opaque hard gel = +ve results. Means tested sample has pyrogen
Fig. – 4 Comparison between Gel Clot Test and Chromogenic Test {final +ve results] as a large vol. assay. Demonstrates the hard gel aspect and the clear transparent aspect of the either respectively. This demonstrates the
quality of the technology and the vol., making capacity of the invented process.
Fig. – 5a Crab dissection operation under way, with the participation of a multi-disciplinary team. Inventor is 2nd from right.
Fig. – 5b Operated adult crab. Shows high fecundity {hundred of eggs} AND paired oviducts.
Fig. – 6 The blue blood. Arrow head points to the granulocytes. Matured size being between 17-22μmt. The granules are as alike human platelets.
Fig. – 7 Under water image of a adult Limulus Polyphemus. The animal always tip-toes
Fig. – 8 Under water image of a adult Tachypleus Tridentus {front member} and a the Tachypleus Gigas {hind member}. Identifications are
tentative.
Fig.-10. Presents the morphological characteristics of the globally well known 4 species of the horse shoe crabs [ ref 12 ].
Source of support: Nil, Conflict of interest: None Declared
